CN106990048A - A kind of work method of detecting bacterium based on photoacoustic imaging technology - Google Patents
A kind of work method of detecting bacterium based on photoacoustic imaging technology Download PDFInfo
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Abstract
A kind of work method of detecting bacterium based on photoacoustic imaging technology, is related to Bacteria Detection living.There is provided and can be used for detection single bacterium or two kinds of mixed cells, quick, easily detection probiotics and pathogenic bacteria relative concentrations treat a kind of work method of detecting bacterium based on photoacoustic imaging technology of offer for providing important references value for clinical guidance.Method one：Photoacoustic imaging reinforcing agent is added and is incubated in the nutrient solution of bacterium living, bacterial solution is swallowed to obtain to probe；Gained bacterial solution is taken into 150 μ L, adds volume in 200 μ L new EP pipes, to carry out the work method of detecting bacterium based on photoacoustic imaging technology.Method two：Two kinds of different photoacoustic imaging reinforcing agents are separately added into the nutrient solution of probiotics and pathogenic bacteria and are incubated, in order to ensure bacterial action and promote phagocytosis of the bacterium to probe；The work method of detecting bacterium based on photoacoustic imaging technology is carried out, according to gained optoacoustic intensity level, the relative concentration ratio of probiotics and pathogenic bacteria is calculated.
Description
Technical field
The present invention relates to Bacteria Detection living, more particularly, to a kind of work method of detecting bacterium based on photoacoustic imaging technology.
Background technology
Tiny ecosystem is the important composition composition of human intestine's system.And the overwhelming majority is in the microorganism in human body intestinal canal
The bacterium that survives of the number in terms of 100,000,000.The bacterium of these huge numbers can substantially be divided into three major classes：Probiotics, pathogenic bacteria
With neutral bacterium.The balance of the stable state of flora, especially probiotics and pathogenic bacteria, maintains the health of human body.Probiotics is mainly wrapped
Include：Lactobacillus, clostridium butyricum, Bifidobacterium, enterococcus faecalis, VREF, actinomyces, saccharomycete etc., and the pathogenic bacteria of human body
Mainly include：Enteropathogenic Escherichia coli, salmonella, Shigella, proteus, bacteroides fragilis, Yersinia etc..
Host-microorganism symbiosis is present, and about 10 are included in the intestines and stomach of each human individual13~1014Individual microorganism
Body, averagely there are about 600,000 kind of microbial gene, and relative abundance has certain individual difference between different Pseudomonas.Gut flora
Diversity influenceed by factors such as heredity, diet, age, environment, area differentiation and antibiotic usages, it is many when enteric bacteria
Sample, which declines, can destroy the stability of flora.Alimentary canal Tiny ecosystem and colorectal cancer and Process of Malignant Transformation of Colon Adenoma are closely related.Knot is straight
Enteric microorganism in the mucous membrane tissue or excrement of intestinal cancer, adenomatous polyp patient and healthy population, which is constituted, to be compared point
Analysis, finds microbial diversity and dominant microflora reduction.Enteric microorganism makes intestinal mucosa proinflammatory signal transduction mechanism
It is abnormal, cause the reparation of intestinal mucosa epithelial damage to aggravate, in addition some enteric microorganism and its in metabolite to intestinal mucosa
Epithelial cell has direct cytotoxic effect, and the incomplete reparation of damaged intestine mucosal epithelium ultimately results in Colon and rectum and swollen
The formation and canceration of knurl.
With continuing to develop for metagenomics, high-flux sequence and bioinformatic analysis technology, the mankind are to microorganism
It is more and more accurate that effect of the group in health and disease recognizes.Although there is the high advantage of accuracy in these detection techniques,
But time-consuming long, cost height, it is impossible to the real-time status of detection bacterium living, Clinical practicability relative deficiency.Thus, research and develop it is low into
Originally, simple and easy to operate, quick detection work bacterium method, is significant.
Bibliography：
1.Nie L.and Chen X.,Structural and functional photoacoustic molecular
tomography aided by emerging contrast agents.Chem Soc Rev,2014.43(20):p.7132-
70。
2.Liu Y.,Nie L.and Chen X.,Photoacoustic Molecular Imaging:From
Multiscale Biomedical Applications Towards Early-Stage Theranostics.Trends
Biotechnol,2016.34(5):p.420-33。
3.Ren Y.D.,et al.,Fecal Microbiota Transplantation Induces HBeAg
Clearance in Patients with Positive HBeAg after Long-Term Antiviral
Therapy.Hepatology,2016[Epub ahead of print]。
The content of the invention
It is an object of the invention to provide available for detection single bacterium or two kinds of mixed cells, quickly, easily detect
Probiotics and the relative concentration of pathogenic bacteria, the offer one kind for providing important references value is treated for clinical guidance based on photoacoustic imaging
The work method of detecting bacterium of technology.
One of work method of detecting bacterium of the present invention based on photoacoustic imaging technology, comprises the following steps：
1) it is incubated in the nutrient solution that photoacoustic imaging reinforcing agent is added to bacterium living, in order to ensure bacterial action and promote
Phagocytosis of the bacterium to probe, obtains bacterial solution；
In step 1) in, the photoacoustic imaging reinforcing agent may be selected from indocyanine green, and the concentration of the indocyanine green can be
0.4mg/ μ L, absworption peak is 790nm wavelength, within the scope of 680~980nm of photoacoustic imaging instrument optimum wavelength, and to bacterium
Without lethal effect, the photoacoustic imaging instrument can use the photoacoustic imaging systems of U.S. Endra Nexus 128；The temperature of the phagocytosis
Can be 37 DEG C, the rotating speed of phagocytosis can be 200rpm, and the time of phagocytosis can be 8~12h.
2) gained bacterial solution is taken into 150 μ L, adds volume in 200 μ L new EP pipes, to carry out being based on photoacoustic imaging skill
The work method of detecting bacterium of art.
The two of work method of detecting bacterium of the present invention based on photoacoustic imaging technology, comprise the following steps：
1) two kinds of different photoacoustic imaging reinforcing agents are separately added into the nutrient solution of probiotics and pathogenic bacteria and are incubated,
In order to ensure bacterial action and promote phagocytosis of the bacterium to probe, probiotics is lactobacillus, clostridium butyricum, Bifidobacterium, excrement intestines
One kind in coccus, VREF, actinomyces, saccharomycete etc.；Pathogenic bacteria are EHEC, salmonella, Shigella, change
One kind in shape bacillus, bacteroides fragilis, Yersinia etc.；
In step 1) in, the photoacoustic imaging reinforcing agent can be Prussian blue, and the Prussian blue concentration is 0.4mg/ μ
L, absworption peak is 713nm wavelength, within the scope of 680~980nm of photoacoustic imaging instrument optimum wavelength, and bacterium is made without killing
With the photoacoustic imaging instrument can use the photoacoustic imaging systems of U.S. Endra Nexus 128；The temperature of the phagocytosis can be 37 DEG C,
The rotating speed of phagocytosis can be 200rpm, and the time of phagocytosis can be 8~12h.
2) 75 μ L are respectively taken to mix gained probiotic bacteria solution and pathogenic bacteria bacterial solution, it is 200 μ L's to add volume
In new EP pipes, the work method of detecting bacterium based on photoacoustic imaging technology is carried out, according to gained optoacoustic intensity level, probiotics is calculated
With the relative concentration ratio of pathogenic bacteria.
In step 2) in, the calculation formula of the relative concentration ratio for calculating probiotics and pathogenic bacteria is as follows：
Wherein, E is photoacoustic signal value of the pathogenic bacteria at different wave length；F is optoacoustic letter of the probiotics at different wave length
Number value；G is the photoacoustic signal value of the mixed bacteria liquid of Escherichia coli and Bacillus acidi lactici respectively at 800nm and 720nm different wave lengths,
Indicate respectively indocyanine green and Prussian blue marked work bacterium；C is the concentration of different bacterium.
The present invention is by Bacterial stain culture living, the work Bacteria Detection based on photoacoustic imaging technology, and bacterium living is trained
Support and dye, the method that Bacteria Detection is carried out using photoacoustic imaging technology can be used for the quick photoacoustic imaging of single work bacterium
Detection, it can also be used to the mixing relative concentration identification of two kinds of bacteriums living.Advantage of the invention is that viable bacteria can be directly used in inspection
Survey, with it is simple to operate, detect the clear superiority such as quick, radiationless, inexpensive.
Brief description of the drawings
Fig. 1 inhales for single work bacterium (Bacillus acidi lactici), reinforcing agent (indocyanine green) and solvent based on photoacoustic imaging technology
Receive peak figure.In Fig. 1, curve a is Lac, and b is Lac-ICG, and c is Medium.
Fig. 2 inhales for single work bacterium (Escherichia coli), reinforcing agent (Prussian blue) and solvent based on photoacoustic imaging technology
Receive peak figure.In fig. 2, curve a is E.coli, and b is E.coli-PB, and c is Medium.
Fig. 3 is single work bacterium (Bacillus acidi lactici) testing result figure based on photoacoustic imaging technology.
Fig. 4 is single work bacterium (Escherichia coli) testing result figure based on photoacoustic imaging technology.
Fig. 5 is two kinds of bacterium (720nm wavelength) hybrid detection result figures living based on photoacoustic imaging technology.
Fig. 6 is two kinds of bacterium (800nm wavelength) hybrid detection result figures living based on photoacoustic imaging technology.
Fig. 7 is the single work Bacteria Detection result figure based on photoacoustic imaging technology.
Embodiment
Embodiment 1：
Referring to Fig. 1~4, a kind of single work method of detecting bacterium based on photoacoustic imaging technology, step is as follows：
(1) single work Bacterial stain culture
Photoacoustic imaging reinforcing agent is added and is incubated in the nutrient solution containing bacterium living, inoculum is according to different bacterium
The growth conditions of strain selects most suitable nutrient solution.In order to ensure bacterial action and promote phagocytosis of the bacterium to probe, using temperature 37
DEG C, rotating speed 200rmp, 8~12h of time.Optoacoustic reinforcing agent one is：Indocyanine green, its concentration is 0.4mg/mL.It is characterized in that
Absworption peak is 790nm wavelength, within the scope of 680~980nm of photoacoustic imaging instrument optimum wavelength, and bacterium is made without killing
With.Sound reinforcing agent two is：Prussian blue, its concentration is 0.4mg/mL.It is characterized in that absworption peak is 713nm wavelength, in optoacoustic
Within the scope of 680~980nm of Image-forming instrument optimum wavelength, and to bacterium without lethal effect.
(2) above-mentioned gained bacterial solution is taken into 150 μ L, added in the new EP pipes that volume is 200 micro- lifes, using the U.S.
The photoacoustic imaging systems of Endra Nexus 128 carry out photoacoustic imaging detection.
Embodiment 2：
Referring to Fig. 5~7, a kind of two kinds of bacterium mixing detection methods living based on photoacoustic imaging technology, step is as follows：
(1) single work bacterium dyes culture respectively
Two kinds of different photoacoustic imaging reinforcing agents are separately added into the nutrient solution containing profitable probliotics or pathogenic bacteria and incubated
Educate, the growth conditions according to different strains selects most suitable nutrient solution.In order to ensure bacterial action and promote bacterium to gulp down probe
Bite, using 37 DEG C of temperature, rotating speed 200rmp, 8~12h of time.Optoacoustic reinforcing agent one is：Indocyanine green, its concentration is 0.4mg/
mL.It is characterized in that absworption peak is 790nm wavelength, within the scope of 680~980nm of photoacoustic imaging instrument optimum wavelength, and it is right
Bacterium is without lethal effect.Sound reinforcing agent two is：Prussian blue, its concentration is 0.4mg/mL.It is characterized in that absworption peak is 713nm
Wavelength, within the scope of 680~980nm of photoacoustic imaging instrument optimum wavelength, and to bacterium without lethal effect.
(2) 75 μ L are respectively taken to mix above-mentioned gained probiotic bacteria solution and pathogenic bacteria bacterial solution, it is 200 to add volume
In μ L new EP pipes, photoacoustic imaging detection is carried out using the photoacoustic imaging systems of U.S. Endra Nexus 128.Probiotics is newborn bar
Bacterium, clostridium butyricum, Bifidobacterium, enterococcus faecalis, VREF, actinomyces, saccharomycete etc..Pathogenic bacteria are EHEC, sand
Door Salmonella, Shigella, proteus, bacteroides fragilis, Yersinia etc..
(3) according to gained optoacoustic intensity level, the relative concentration ratio of probiotics and pathogenic bacteria is calculated.
Calculation formula is as follows：
Wherein, E is photoacoustic signal value of the pathogenic bacteria at different wave length；F is optoacoustic letter of the probiotics at different wave length
Number value；G is the photoacoustic signal value of the mixed bacteria liquid of Escherichia coli and Bacillus acidi lactici respectively at 800nm and 720nm different wave lengths,
Indicate respectively indocyanine green and Prussian blue marked work bacterium；C is the concentration of different bacterium.
It is contemplated that it is high to improve the time-consuming long, cost of the existing detection technique for human body bacterium, it is impossible to detection bacterium living
The limitation such as real-time status.Advantage of the invention is that it is inexpensive, simple and easy to operate, can quick detection live bacterium, it is clinical real
It is relatively large with property, it is significant.
Claims (10)
1. one of a kind of work method of detecting bacterium based on photoacoustic imaging technology, it is characterised in that comprise the following steps：
1) it is incubated in the nutrient solution that photoacoustic imaging reinforcing agent is added to bacterium living, in order to ensure bacterial action and promote bacterium
Phagocytosis to probe, obtains bacterial solution；
2) gained bacterial solution is taken into 150 μ L, adds volume in 200 μ L new EP pipes, to carry out based on photoacoustic imaging technology
Method of detecting bacterium living.
2. one of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 1, it is characterised in that in step
It is rapid 1) in, the photoacoustic imaging reinforcing agent be selected from indocyanine green.
3. one of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 2, it is characterised in that described
The concentration of indocyanine green is 0.4mg/ μ L, and absworption peak is 790nm wavelength, in 680~980nm of wavelength of photoacoustic imaging instrument.
4. one of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 4, it is characterised in that described
Photoacoustic imaging instrument uses the photoacoustic imaging systems of U.S. Endra Nexus 128.
5. one of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 1, it is characterised in that in step
It is rapid 1) in, 37 DEG C of the temperature of the phagocytosis, the rotating speed of phagocytosis is 200rpm, and the time of phagocytosis is 8~12h.
6. the two of a kind of work method of detecting bacterium based on photoacoustic imaging technology, it is characterised in that comprise the following steps：
1) two kinds of different photoacoustic imaging reinforcing agents are separately added into the nutrient solution of probiotics and pathogenic bacteria and be incubated, in order to
Ensure that bacterial action simultaneously promotes phagocytosis of the bacterium to probe, probiotics be lactobacillus, clostridium butyricum, Bifidobacterium, enterococcus faecalis,
One kind in VREF, actinomyces, saccharomycete；Pathogenic bacteria be EHEC, salmonella, Shigella, proteus,
One kind in bacteroides fragilis, Yersinia；
2) respectively take 75 μ L to mix gained probiotic bacteria solution and pathogenic bacteria bacterial solution, add the new EP that volume is 200 μ L
Guan Zhong, carries out the work method of detecting bacterium based on photoacoustic imaging technology, according to gained optoacoustic intensity level, calculates probiotics and cause
The relative concentration ratio of germ.
7. the two of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 6, it is characterised in that in step
It is rapid 1) in, the photoacoustic imaging reinforcing agent is Prussian blue.
8. the two of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 7, it is characterised in that described
Prussian blue concentration is 0.4mg/ μ L, and absworption peak is 713nm wavelength, in 680~980nm of photoacoustic imaging instrument wavelength, described
Photoacoustic imaging instrument uses the photoacoustic imaging systems of U.S. Endra Nexus 128.
9. the two of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 6, it is characterised in that in step
It is rapid 1) in, 37 DEG C of the temperature of the phagocytosis, the rotating speed of phagocytosis is 200rpm, and the time of phagocytosis is 8~12h.
10. the two of a kind of work method of detecting bacterium based on photoacoustic imaging technology as claimed in claim 6, it is characterised in that in step
It is rapid 2) in, the calculation formula of the relative concentration ratio for calculating probiotics and pathogenic bacteria is as follows：
Wherein, E is photoacoustic signal value of the pathogenic bacteria at different wave length；F is photoacoustic signal value of the probiotics at different wave length；
G is the photoacoustic signal value of the mixed bacteria liquid of Escherichia coli and Bacillus acidi lactici respectively at 800nm and 720nm different wave lengths, respectively
Indicate indocyanine green and Prussian blue marked work bacterium；C is the concentration of different bacterium.
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Cited By (2)
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| CN108469415A (en) * | 2018-03-14 | 2018-08-31 | 上海交通大学 | Liquid trace concentration detection method and device based on nanogold particle enhancing |
| CN115671361A (en) * | 2022-11-07 | 2023-02-03 | 浙江理工大学 | Preparation method of diagnosis and treatment type core-shell nanofiber membrane |
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| CN108469415A (en) * | 2018-03-14 | 2018-08-31 | 上海交通大学 | Liquid trace concentration detection method and device based on nanogold particle enhancing |
| CN115671361A (en) * | 2022-11-07 | 2023-02-03 | 浙江理工大学 | Preparation method of diagnosis and treatment type core-shell nanofiber membrane |
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